As the demand of constructing Illumina libraries increases, we have started to modify the library construction protocol to adapt the use of multichannel pipette and 96-well plates. With the few simple modification steps, we have doubled the library production efficiency. These modifications include the shearing of DNA with Covaris E210, and the cleaning of enzymatic reactions and fragment size selection with SPRI beads and a magnetic plate holder. We have also designed a set of molecular barcodes to enable the sequencing of many libraries in parallel. The requirements of these barcodes include 4 bases, balanced GC, and at least 2 bases difference between barcodes. The barcode is attached to the adaptor so it does not require third sequencing primer and the barcoded library can be run on the same flowcell/run with other non-barcoded libraries. We have begun to assess the ability to assign reads and the potential bias towards certain barcodes after pooling different number of libraries. We have recently programmed the Biomek FX robot to carry out the library construction process. Although this process still require manual transfer of plates from robot to other work stations, the processing of 96 Illumina libraries takes approximately 6-8 hours. This semi-automated process represents a significant increase of library capacity comparing to the manual process. We will present the progress and the challenges of these scale-up processes.
At JGI, major efforts have been spent on using the 16S RNA sequencing to study the composition of bacterial community. Currently, 16S rRNA genes from a microbial community are amplified with conserved PCR primers and the mixed amplicon is cloned and sequenced using traditional Sanger sequencing method. Using Roche GS FLX amplicon sequencing method, individual amplicon can be sequenced directly on the platform, producing hundreds of thousand ~;250 bp tags of the full length 16S rRNA gene. We call this pipeline PyroTag. The benefit of the PyroTag technology includes higher resolution and less bias than traditional PCR and cloning based sequencing. The disadvantage is that the read length is short as we used FLX reagent kit for the study. The aim for this study is to systematically test the effect of amplicon length and 16S rRNA regions targeted on diversity coverage of complex microbial community. The goal is to develop a standard PyroTag pipeline to provide to broad range JGI users. Molecular barcode method has been used to distinguish different amplicon length and different targeted 16S regions. Strategy and detailed experiment design as well as preliminary results will be presented.
Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. 3 It is a major degrader of plant cell walls and has been used as a model organism for the study of 4 secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a 5 single circular chromosome of 3642249 bp predicted to encode 3117 proteins and 65 RNA6 species with a coding density of 85 percent. Genome analysis revealed the existence of 29 putative 7 glycoside hydrolases in addition to the previously identified cellulases and xylanases. The 8 glycosyl hydrolases include enzymes predicted to exhibit mainly dextran/starch and xylan 9 degrading functions. T. fusca possesses two protein secretion systems: the sec general secretion 10 system and the twin-arginine translocation system. Several of the secreted cellulases have 11 sequence signatures indicating their secretion may be mediated by the twin-arginine12 translocation system. T. fusca has extensive transport systems for import of carbohydrates 13 coupled to transcriptional regulators controlling the expression of the transporters and14 glycosylhydrolases. In addition to providing an overview of the physiology of a soil 15 actinomycete, this study presents insights on the transcriptional regulation and secretion of16 cellulases which may facilitate the industrial exploitation of these systems.
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